BOD Analysis using LCK555 – brief user guide
DR2800/ DR5000 and:
LZC901 Dilution Water Set,
LZC555 Inoculation Material,
LCA555 BOD Standard
1. Preparation of Dilution Water

Set up the dilution water kit as shown in the diagram. The pump should be placed above the
water level in the jar



Add 500ml tap water to the jar and aerate for 5 minutes to expel free chlorine
Pipette 0.3ml of LCK555 trace element “D” into the jar.
Add appropriate inoculation material to the jar. Either:
i.
ii.
5ml particle-free, municipal wastewater with COD @250mg/l or
20ml particle-free, municipal wastewater with COD @50mg/l
Then aerate for 3 days. This water could be kept for up to 8 days
Or
iii.
0.5ml prepared LZC555 inoculation solution (see box below).
Then aerate for 1 hour. This water should be used immediately.
Preparation of LZC555
1. Add a level dosing spoon of inoculation material into the reaction tube
2. Pipette 10ml of buffer solution into the tube
3. Shake for 1 minute
4. Leave to stand for 1 hour at room temperature
5. Pipette 0.5ml of the solution into the dilution water jar
2. Sample Preparation



Homogenise the sample at 20°C, 700-900rpm for 5 minutes (for larger particulates,
homogenise @20,000rpm for 5 seconds)
Select the appropriate range for the test, based upon estimated BOD. This will determine
volume of sample/ dilution water used in the sample preparation step, and then the dilution
factor entered in the evaluation step after 5 days.
If it is not possible to estimate the BOD, make an approximation from the COD value:





Untreated industrial and municipal wastewater- BOD (mg/l) is @35-65% COD
Biologically treated wastewater- BOD (mg/l) is @25% COD
Prepare the sample in the dilution tube according to the table.
Shake the dilution tube for 1 minute to aerate the sample fully
Transfer the appropriate volume into a cuvette and fill to the brim with dilution water
eg. For a sample with estimated BOD of 75mg/l – measuring range would be B1
Pipette 1ml of sample and 1 ml of dilution water into the dilution tube. Shake, then pipette 0.5ml
into the cuvette and fill with dilution water. Enter dilution factor of 25 in evaluation step




Screw on the cuvette cap, ensuring no air bubbles are present
At the same time as preparing the samples, fill a cuvette with dilution water only. This will be
used as the blank in the evaluation step (1 needed per day’s BOD analysis)
Incubate the cuvettes at 20°C in the dark for 5 days using an appropriate incubator (such as
LT20)
After 5 days, evaluate using DR2800/ DR5000 etc
3. Evaluation using DR2800/ DR5000









Remove the cuvettes from the incubator
For each cuvette in turn remove the foil from the dosi-cap, unscrew the cap and mount the
funnel
Pour in the contents of the dosi-cap (reagent and glass beads) into the cuvette, screw on the
cap (again ensuring no air bubbles present) and invert for 3 minutes
o nb if liquid meniscus drops, add 2-4 glass beads to make up the volume
Wait for a further 3 minutes
Insert the blank into the DR2800/ DR5000
Upon obtaining the E1 message, insert the first sample.
When prompted, enter the appropriate dilution factor for the sample
The instrument will display the result in mg/l
Repeat for each sample cuvette
3. Use of LCA555 for AQC (Analytical Quality Control)









Pipette 5ml distilled water into one of the LCA555 cuvettes
Shake vigorously (upto 3 minutes) to dissolve all of the contents
Pipette 2.2ml of this into a clean 50ml volumetric flask
Top up with distilled water and shake
This is equivalent to a 205mg/l +/-45mg/l BOD standard
Using LCK555, this is analysed in measuring range B2
Pipette 0.5ml of this directly into a cuvette and fill with dilution water
For evaluation, enter a dilution factor of 50
The result should be between 160- 250mg/l
n.b BOD standards can also be prepared using dried glucose/ glutamic acid
Note on use of LCK554 - measuring range 0.5- 12mg/l
Undiluted – 0.5- 6.0mg/l
Diluted (x2) – 1.0- 12.0mg/l








Sample preparation: Pour appropriate sample quantity (undiluted – 40ml sample; Diluted –
20ml sample and 20ml chlorine-free drinking water) into a beaker and stir for 5 minutes at
500- 750rpm
LCK554 is measured directly - 2 sample cuvettes are required for each sample – cuvette 1 is
measured on day 1, cuvette 2 measured on day 5. Label these e.g. A1 and A5
Fill each cuvette to the brim with the prepared sample, and incubate the cuvette 2 sample(s)
for 5 days at 20°C (ensure no air bubbles are present)
For sample 1 cuvette(s), add the dosi cap contents, invert for 3 minutes and wait 3 minutes
Evaluate using spectrophotometer, noting the results for use to calculate the BOD on day 5
On day 5, evaluate the cuvette 2 sample(s) in the same way
Calculate BOD using the day 1 and day 5 results: e.g. A1- A5= BOD (mg/l)
If a dilution is made, ensure this is included in the calculation
Points to Note




Peroxides, strong oxidising or reducing agents and high chlorine concentrations can interfere
with oxidation, resulting in high or low bias
Sample pH should be between 4 and 10
It is important to check the table of interferences in the procedure for ions present in the
sample
It is recommended to run AQC checks, such as with LCA555, every 10 th sample